Smart delivery systems for microbial biofilm therapy: Dissecting design, drug release and toxicological features.

Bacterial biofilms are highly protected surface attached communities of bacteria that typically cause chronic infections. To address their recalcitrance to antibiotics and minimise side effects of current therapies, smart drug carriers are being explored as promising platforms for antimicrobials. Herein, we briefly summarize recent efforts and considerations that have been applied in the design of these smart carriers. We guide readers on a journey on how they can leverage the inherent biofilm microenvironment, external stimuli, or combine both types of stimuli in a predictable manner. The specific carrier features that are responsible for their 'on-demand' properties are detailed and their impact on antibiofilm property are further discussed. Moreover, an analysis on the impact of such features on drug release profiles is provided. Since nanotechnology represents a significant slice of the drug delivery pie, some insights on the potential toxicity are also depicted. We hope that this review inspires researchers to use their knowledge and creativity to design responsive systems that can eradicate biofilm infections.

Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins.

Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. SIGNIFICANCE: Enterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

Study of Adherence Pattern of Antidepressants in Patients with Depression.

Background Depression is one of the leading psychiatric disorders of the world affecting a person's mood, physical health and behavior. It is not permanent and is neither a character flaw nor a lack in discipline for a person to be ashamed of. It is a disorder that is reliably diagnosed and successfully treated. Antidepressants are the standard and the most efficacious approach to treating people with depression. However, adherence to treatment is necessary for achieving effectiveness. The result of nonadherence is severe and may cause therapeutic failure resulting in poor quality of life. Objective To determine the medication adherence pattern in patients with depression and assess the factors associated with non-adherence to the prescribed antidepressant therapy. Method Patients meeting the inclusion criteria who were diagnosed with depression were taken for the study. Informed consent was taken from the patients or from their relatives in case of their incapability. They were then interviewed using structured questionnaire. Result Among the 60 patients included in the study, 78% of them were females. Most of the patients 43% (n=26) were prescribed with atypical antidepressants. Less number (37%) of the patients were adherent to the antidepressant therapy. 68% of the females were non adherent. 82% of housewives were not adherent to therapy. Forgetfulness was the main reason for missing dose in majority (50%) of the nonadherent patients. Conclusion Majority of the patients with depression were non-adherent to medication. Forgetfulness was the major reason for missing dose in these patients.

A Prospective Study of Commonly Prescribed Drugs in the Management of Neuropathic Pain and its Medication Adherence Pattern.

Background Neuropathic pain is one of the common complains of patients visiting neurology and orthopedic departments in hospitals. Management of neuropathic pain is difficult and is often symptomatic rather than being curative. Adherence to medication is necessary for pain management to be effective. However, there are various factors related to patient, physician, drug regimen and other socio-economic affecting adherence. Objective To study commonly prescribed drugs in neuropathic pain management and the medication adherence pattern including its associated factors. Method Patients already diagnosed as neuropathic pain were interviewed using structured questionnaire and data entered in Microsoft Office Excel 2007. Informed consent was taken from the patients. Result Among the 84 patients in the study, 69% were females. Majority 53.6% of patients had low back pain as cause of neuropathic pain. Anticonvulsants were mostly prescribed (75%) followed by non-steroidal anti-inflammatory drugs (52.4%) and Methylcobalamin (47.6%). More than 50% (n=49) patients were not adherent to the prescribed medication and majority (61.2%) of them were housewives. Significant association was observed between patient's adherence to gender, occupation, polypharmacy, drug regimen, cost and availability of medicine. Conclusion Anticonvulsants were commonly prescribed drugs in patients with neuropathic pain. Neuropathic pain was seen more in females with low back pain. Majority of patients were non-adherent and forgetfulness was the major reason for missing dose in them.

Physiological barriers to the oral delivery of curcumin.

Curcumin, a principal component from Curcuma longa, with antioxidant and anti-inflammatory activities was proposed as a potential candidate for the preventation and/or treatment of cancer and chronic diseases. However, curcumin could not achieve its expected therapeutic outcome in clinical trials due to its low solubility and poor bioavailability. The actual intestinal physiological barriers limiting curcumin absorption after oral administration have not been fully investigated. To identify the main barriers curtailing its absorption, in vitro permeability of curcumin and flux of its glucuronide were monitored in rat jejunum and Transwell grown Caco-2 cells. Curcumin was more permeable under acidic conditions, but the permeability was substantially below the permeability of highly permeable standards. Its efflux could not be inhibited by specific Pgp and MRP inhibitors. BCRP was found to participate in curcumin transport, but the Organic Anion Transporting Polypeptide (OATP) did not. The permeability of curcumin significantly increased when the structure of mucus was compromised. The inhibitor of curcumin metabolism, piperin, failed to act as a permeability enhancer. Piperin inhibited Pgp and MRP transporters and decreased the amount of glucuronide transported back into the intestine. Inclusion of piperin in curcumin-containing formulations is highly recommended as to inhibit curcumin glucuronidation and to increase the transport of formed glucuronides into the plasma, therefore increasing the probability of glucuronide distribution into target tissue and inter-convertion to curcumin. It would also be beneficial, if curcumin delivery systems could reversibly compromise the mucous integrity to minimize the non-specific binding of curcumin to its constituents.

Kinetics of cellular and humoral immunity in a successful case of positive crossmatch kidney transplantation: a case report.

A positive crossmatch remains one of the major barriers to successful kidney transplantation. Highly sensitized patients are at greater risk of hyperacute rejection and subsequent graft loss after transplantation. Although recent advances in desensitization therapy allow kidney transplantation in these patients, the success rate is quite low. Herein, we have reported a successful case of positive crossmatch living donor kidney transplantation using a desensitization protocol with an immune monitoring assay. A 42-year-old woman with end-stage renal disease due to IgA nephropathy had been on hemodialysis for 36 months. She showed positive T-cell and B-cell cytotoxic crossmatches with her husband owing to pretransplantation blood transfusions. We performed a preconditioning regimen comprising a single dose of rituximab (375 mg/m(2)) combined with double-filtration plasmapheresis (DFPP) followed by low doses of intravenous immunoglobulin (DFPP/IVIG treatment). Tacrolimus (target trough level, 5-10 ng/mL) and mycophenolate mofetil (1500 mg/body) were started 2 weeks before the DFPP/IVIG treatment. After 6 DFPP/IVIG sessions, the crossmatch became negative. An induction quadruple immunosuppression protocol included tacrolimus, mycophenolate mofetil, basiliximab, and methylprednisolone. After the transplantation, the patient's immune status was evaluated regularly by mixed lymphocyte reactions (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique (CFSE-MLR assay) and immunosuppressant therapy was adjusted accordingly. During the observation period, neither antibody-mediated rejection nor acute cellular rejection was encountered in this patient.

The influence of ethnic origin on the skin photoageing: Nepalese study.

Ethnic and genetic differences modify skin structure and function. Skin photoageing is becoming one of the most studied cosmetological topics. However, there are relatively few data available to evaluate the effect of ethnic skin origin on the degree of photoageing, particularly among people inhabiting sunny and hot climate as in South Asia. Two hundred and forty participants, enrolled in the study conducted in Pokhara valley, Nepal, were classified into four different age categories viz. below 30, 30-50, 50-60 and above 60 years. Participants answered the questioner regarding their age, gender, ethnic origin, occupation, skin characteristics, lifestyle factors, type of cosmetic and skin care products used, drug treatment and personal skin condition evaluation. Skin hyperpigmentation and the degree of wrinkling, the two most important parameters related to photoageing, were particularly evaluated in two ethnic groups, namely Aryan-origin and Mongolian-origin participants. The study showed that Aryan-origin ethnic skin was more wrinkled and darker than that of Mongolian. Male skin was found to be darker than the female skin, which might be the result of the use of sun-protective cosmetic products among women. Smoking was found to increase the degree of wrinkling; however, no differences related to ethnic origin in relation to smoking were observed. This study indicates the need to further evaluate the differences in skin response to daily exposure to strong sun among various ethnic-origin inhabitants.

Need of improvement in timing of prophylactic antibiotic in elective surgery.

INTRODUCTION: Infections in surgery are major concern of morbidity, mortality, and costs. Timely antibiotic prophylaxis before incision ensures optimum concentration of AP in blood and tissues to prevent surgical site infections . However, proper timing of AP remains problematic as reported by various studies, though none so far from Nepal. Aim of this prospective observational study was to assess and address the issues for improvements in timing of AP. METHODS: The pattern of antibiotic prophylaxis were prospectively collected in respect to time of induction, and incision time were recorded on predesigned 'AP form'. The study included all the elective major cases who received AP of intravenous Cefazoline 1 g as per our existing protocol. The emergency surgeries and obstetric cases were excluded from the study. RESULTS: There were 125 cases of which 89% received AP before incision (63% within 5 minutes before incision), while 11% had AP after the incision and 1% within the recommended time period of 2 hour to 30 minutes before incision. CONCLUSIONS: Current practice of antibiotic prophylaxis (AP) needs improvement as per standard guidelines of AP within 2 hour to 30 before incision.

Screening of Nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition.

South-East Asian population is daily exposed to strong sunlight. As a result, the majority of population will have darker, ethnic skin. Moreover, many people suffer from dark spots, hyperpigmentation, which is considered to be a skin disorder and causes psychological disturbance. To treat dark spots, most of the population will still rely on traditionally used crude drugs, knowledge about which is transferred from generation to generation. Fifty-two crude drugs were selected based on the survey performed among local healers and beauticians of different ethnic origin. These crude drugs were screened for mushroom tyrosinase inhibitory activity, as tyrosinase inhibitors are becoming increasingly important as cosmetic and medicinal products, primarily to control hyperpigmentation. Among the tested crude drugs, methanolic extracts of Glycyrrhiza glabra, Morus alba, Syzygium aromaticum, Citrus aurantifolia, Cypreae moneta, Punica granatum and Citrus aurantium, at the final concentration of 50 microg mL(-1), showed mushroom tyrosinase inhibitory activity of 78.9%, 71.0%, 69.4%, 59.0%, 56.0%, 53.4 and 51.9%, respectively, with 91.4% inhibitory activity of kojic acid taken as positive control. To our knowledge, this is the first report that extracts of Cypreae moneta shell and Syzygium aromaticum flowering bud have tyrosinase inhibitory activity. These potent extracts were further evaluated at different concentration. The final concentration of the extracts in reaction mixtures was 50, 25 and 5 microg mL(-1) for the initial concentration of 1000, 500 and 100 microg mL(-1), respectively. They showed concentration-dependent inhibition of mushroom tyrosinase. Those extracts expressing relatively weak tyrosinase inhibitory activity may act through different inhibition pathway which is not based on tyrosinase activity. Further evaluation of the most potent tyrosinase inhibitors in in vivo conditions would be recommended.

The efficacy and safety of low dose epidural butorphanol on postoperative analgesia following cesarean delivery.

Butorphanol is considered an effective and safe analgesic after cesarean delivery but is associated with profound dose-dependent sedation. Somnolence may cause hindrance in early mother-baby interaction. This study was designed to assess the analgesic efficacy and to monitor side-effects of low doses (0.5 mg and 0.75 mg) of epidural butorphanol with bupivacaine compared to bupivacaine alone in parturients following cesarean delivery. One hundred and twenty parturients (American Society of Anesthesiologists physical status 1 and 2) undergoing cesarean delivery were allocated into three groups: group 1 received epidural 0.125% bupivacaine while group 2 and 3 received an additional 0.5 mg and 0.75 mg butorphanol respectively. A combined spinal, epidural technique was used. Spinal anaesthesia was used for surgery. The epidural route was used for postoperative analgesia with the study drug. Onset, duration and quality of analgesia, lowest visual analogue scales (VAS) score, and side effects were noted. The onset and duration of analgesia in group 2 (4.1+/-2.6 min and 202.4+/-62.8 min) and group 3 (4.0+/-2.5 min and 192.3+/-69.1 min) were significantly different (P<0.01) from group 1 (6.6+/-2.7 min and 145.7+/-89.6 min). The quality of analgesia in terms of time to first independent movement and satisfactory VAS were statistically better (P<0.01) in group 2 (3.9+/-0.3 hour and 8.1+/-0.1 mm) and group 3 (3.8+/-0.4 hour and 8.1+/-0.9 mm) than in group 1 (5.2+/-0.4 hour and 6.3+/-1.3 mm). The incidence of sedation was 5% in all the three groups. A lower dose of epidural butorphanol with bupivacaine produces a significantly earlier onset, longer duration and better quality of analgesia than bupivacaine does.

Prevalence of methicillin resistant Staphylococcus aureus in school children of Pokhara.

Present study was carried out to find out the prevalence of methicillin resistant Staphylococcus aureus (MRSA) in school children of Pokhara city in western, Nepal. A total of 184 randomly selected children younger than 15 years were included in the study. Nasal swabs collected were subjected to standard bacteriological culture. S. aureus isolates were identified by mannitol fermentation, coagulase positivity and DNase positivity. Antimicrobial susceptibility test was performed on muller-hinton agar (MHA) by modified Kirby-Bauer disc diffusion method. Out of total 184 nasal swabs, S. aureus was isolated in 31.0% (n=57). Among the isolates, 35.1% (n=20) were from male children whereas 64.9% (n=37) were from female. There was no significant sex difference in colonization of S. aureus. Out of 57 isolates, 56.1% (n=32) were MRSA. MRSA isolates indicated relatively high rate of resistance to antibiotic cloxacillin (68.7%) followed by ofloxacin (40.6%), tetracycline (15.6%), erythromycin (9.4%), ciprofloxacin (6.2%) and vancomycin (3.1%).This study showed a high prevalence of MRSA carriage in school children indicating the spread of MRSA in the community.

Congenital heart disease in Nepalese children.

The present status of congenital heart disease (CHD) from the persepective of the children's health in Nepal is not yeat available. Cross-sectional analysis of all children with heart disease attending Kanti Children Hospital was performed. VSD, ASD, obstructive valvular lesions and cyanotic heart diseases were the most common CHDs. Present data indicate the need for increased levels of cardiac care in children, correctional surgeries, monitoring of the patients and awareness among caregivers of CHD patients.

Tinea capitis infection in school children of Nepal.

From among 428 Nepalese schoolchildren hair samples of 102 children with clinical features of tinea capitis, obtained by the sterile hairbrush method, were examined by mycological techniques. Age varied between 4-16 years. Itching was experienced by 96.1% sample subjects and hair loss by 32.4%. Of the 102, 11 (10.8%) were positive for Trichophyton violaceum (TV), 6 being from urban areas, the rest from rural areas. Amongst the 11 patients, 7 (63.6%) were girls and rest boys. Statistical associations were observed between the place of haircut and isolation of the organism (chi2 = 15.2, p <0.01). Statistical association was also present between frequency of bathing and isolation of organism. Sharing of combs was associated with the culture-positive subjects. The prevalence of tinea capitis in the urban and rural children was 2.3% and 3.0%, respectively. The only isolated organism was TV. An association of the isolation of TV was found with risk factors such as family members, sharing of combs, frequency of bathing with the organism. Hair loss was more common in the urban children. Discouragement of sharing combs, increased frequency of hair washing, and use of uncontaminated hair cutting instruments are recommended.

Liposomal gels for vaginal drug delivery.

The aim of our study was to develop a liposomal drug carrier system, able to provide sustained and controlled release of appropriate drug for local vaginal therapy. To optimise the preparation of liposomes with regards to size and entrapment efficiency, liposomes containing calcein were prepared by five different methods. Two optimal liposomal preparations (proliposomes and polyol dilution liposomes) were tested for their in vitro stability in media that simulate human vaginal conditions (buffer, pH 4.5). To be closer to in vivo application of liposomes and to achieve further improvement of their stability, liposomes were incorporated in vehicles suitable for vaginal self-administration. Gels of polyacrylate were chosen as vehicles for liposomal preparations. Due to their hydrophilic nature and bioadhesive properties, it was possible to achieve an adequate pH value corresponding to physiological conditions as well as desirable viscosity. In vitro release studies of liposomes incorporated in these gels (Carbopol 974P NF or Carbopol 980 NF) confirmed their applicability as a novel drug carrier system in vaginal delivery. Regardless of the gel used, even 24 h after the incubation of liposomal gel in the buffer pH 4.5 more than 80% of the originally entrapped substance was still retained.

Mucoadhesive chitosan-coated liposomes: characteristics and stability.

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280 nm, whereas the size after coating was 300-330 nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65% of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.

Delivery of antisense oligonucleotides to neuroblastoma cells.

pH-Sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) were applied in delivery of antisense oligodeoxynucleotides (asODN) into NG 108-15 neuroblastoma and glioma cells. Fluorescently labelled asODN were entrapped in liposomes by a modified freeze-thawing method (20% encapsulation efficiency). The uptake of asODN (free or entrapped in liposomes) by NG 108-15 cells was monitored by fluorescence-activated cell sorting and confocal microscopy. Delivery of asODN was significantly improved when antisense were entrapped in liposomes as compared to free (nonliposomal) asODN. The uptake was dose-dependent and optimum was achieved after 2 h incubation.

Mycological examination of the hair samples of 11 school-going Nepalese children suspected of tinea capitis.

Among 150 students from a semi-urban school in Kathmandu (Nepal), 30 from various age groups and both sexes with symptoms suggestive of scalp ringworm, were examined clinically. Hair samples were collected for mycological examination, using the hair brush method, from 11 of the 30 children (36.7%), aged between 5 and 14 years, who were clinically diagnosed. Four of the cultured samples were positive for isolates of Trichophyton violaceum. This minor study provides evidence that tinea capitis might constitute a substantial infectious dermatological problem in Nepal. The study also indicated T. violaceum as the causative organism of tinea capitis.